1. Field of the Invention
This invention relates to systems, more particularly a device for quantitative colorimetric analysis of organic substances concentrations generally in biological fluids. The invention also relates to a method for making such analysis. The invention also relates to a system, a device and a method which involves the reduced and oxidized coenzymes nicotinamide-adenine dinucleotide (NADH, NAD+) or nicotinamide-adenine dinucleotide phosphate (NADPH, NADP+) (collectively herein referred to as NAD(P)H and NAD(P)+) in a system, a device and a method for determination of NAD(P)H. The device may be disposable.
The invention relates to a novel and unique analog to digital colorimetric signal system, device and method which determines the concentration of NAD(P)H or of an organic substance which generates NAD(P)H in a NAD(P)+ dependent dehydrogenase reaction where the dehydrogenase is specific to the substrate.
The concentration of the NAD(P)H or of the organic substance of unknown concentration, the concentration which is determined, is ascertained by an "off"-"on" change of color, preferably quite decisive and highly distinctive, like yellow to intense blue.
The invention provides a test of extreme sensitivity and accuracy coupled with great convenience. The invention has numerous applications and uses in industrial, biomedical, medical, diagnostic (e.g., in genetic engineering) and numerous other fields as will become readily apparent to one of average skill in the art to which the invention pertains.
2. Brief Description of the Prior Art
The earlier filed patent application referred to above describes a system for colorimetrically determining the concentration of a biological molecule in an aqueous solution, such as saliva, blood, urine, or an industrial process fluid.
The contribution made by the invention disclosed by the prior application is described below. For a detailed description of the prior art, reference is made to the earlier filed pending application. Briefly, the use of dehydrogenase enzymes as specific probes for biological molecules is well known in the art. In general, a biological molecule to be assayed is oxidized by a substrate-specific dehydrogenase, and the resulting product, NAD(P)H, is either assayed directly or converted into a color signal and assayed. When NAD(P)H is assayed directly, the known fact that NAD(P)H has a substantial difference in absorbance at 340 nm than does NAD(P), is used as a measure of the amount of dehydrogenase substrate oxidized. When the NAD(P)H is converted into a color signal, an enzyme or catalyst is used to transfer the electrons from the NAD(P)H to a chromogenic molecule which accepts these electrons with a resulting change in visible color.
A serious problem that existed in all of the art prior to the earlier application is that one equivalent of dye molecule is produced for ever biological molecule that is oxidized. This limitation prevented the assay of "high" concentrations of these biological molecules, as the amount of color that would be generated by the complete or near complete oxidation of such a "high" concentration, in accordance with conventional methods, would yield colored solutions that had too high an absorbance to be read without dilution. Additionally, the "high" concentration of dye would lead to solutions wherein dye-dye interaction would cause serious deviation from the ideal as predicted by Beer's law, resulting in solutions that do not have linear relationship of absorbance versus concentration of dye. These difficulties are of more than passing concern. These "high" concentrations (too high to measure), include the normal concentration as well as the super normal concentration indicative of a disease state of most, if not all, medically important biological metabolites. Therefore, when complete or near complete oxidation of a biological molecule was used in the assay methodology common to all previous assays, a massive dilution of the medical sample was necessary to lower the concentration of the biological molecule.
The prior patent application overcomes these problems. In the invention described in that patent application less than one molecule of dye is created per molecule of NAD(P)H. This ratio of less than one to one is accomplished in a system that contains a diaphorase enzyme that catalyzes the NAD(P)H dependent reduction of a chromogen to cause a visible color change, a first substrate, which is a chromogenic electron-acceptor and a substrate for the diaphorase, which causes color to be changed when the chromogen is reduced by NAD(P)H; and a competing substrate for the diaphorase which is an electron-acceptor but which does not undergo a colorimetric change in the same region of the visible spectrum as does the chromogenic substrate. The patent application discloses methods for selecting and identifying competing substrates, all such competing substrates being substrates for the diaphorase enzyme.
Of particular note in that invention was the discovery of a class of competing substrates which prevented the generation of color unless a predetermined amount--the threshold amount--of the substance to be measured was present. This embodiment of the invention enables one skilled in the art to produce a unique, colorimetric, analog to digital converter. When the concentration of NAD(P)H is less than the threshold concentration, no color is produced. At any concentration of NAD(P)H greater than the threshold concentration, a dark color is produced. This threshold can be set at any given concentration, or a multitude of regions on a measuring device can have different concentrations, each of which will measure a different concentration as a digital "on/off" signal.